^?.>-.7* 


Circular  No    94. 


United  States  Department  of  \gm 


EAU   OK   KNTOMOI 

i  Bui 


\mi  1:11  w  i  oi  I    i:i:ooi). 


F.  Win  ik,  Ph.  I'.. 
\ii  in  Bactei iolo 

For  Beveral  reasons  much  confusion  exists  ae  to  the  preBenl  statue  oi 
mir  knowledge  of  bee  diseases.  It  is  hoped  thai  this  circular  will  give 
information  which  will  to  Bomeextenl  clear  up  the  subject  ol  American 
foul  brood  from  a  bacteriological  poinl  of  view.  The  Bymptoms  of  this 
re  given  in  Circular  No.  79  of  the  Bureau  of  Entomology, 
entitled  "The  Brood  Diseases  of  Bees."  American  foul  brood  is  the 
prevalent  disease  in  America,  and,  judging  from  reports  received  from 
pe  and  from  descriptions  in  European  bee  journals  and  hooks,  it  is 
the  prevalent  one  there.  Then'  is  another  diseased  condition,  which 
Cheyne  examined,  and  to  which  we  now  refer  as  European  foul  brood. 

When  tlic  author  began  his  work  on  bee  diseases  in  the  Bummer  of 
1902  he  observed,  in  combs  containing  American  foul  brood,  in  the 
dried  remains  of  the  dead  larva',  known  as  the  Bcales,  a  very  large  num- 
ber of  Bpores  which  failed  to  grow  when  inoculated  into  the  media  ordi- 
narily used  in  the  laboratory.  It  was  clear,  then,  that  these  Bpores  are  not 
Bacillus  alvei  and  thai  this  disease  is  not  the  "foul  brood  "  of  Cheshire 
and  Cheyne.  The  follow ing  year  the  study  was  continued  and  a  medium 
levised  in  which  the  Bpores  found  in  this  disease  will  germinate. 
This  medium  consist-  of  an  agar  made  by  following  the  directions  ordi- 
narily used  in  the  laboratory,  with  the  exception  that  bee  larva?  are  sub- 
stituted for  meat.  By  the  use  of  tins  medium  were  obtained  pure  cul- 
tures of  the  microorganism  which  is  found  so  abundantly,  in  the  form 

of  Bpores,  in  the  dried  BCaleS  of  American  foul  hrood. 

In  reporting  these  findings  the  author  referred  to  this  organism  as 

Bacillus    \. "      Further  study  was  subsequently  made,  and  the  -pedes 

was  given  the  permanent  name    Bacillus  larva.    The  description  of 

this  species  may  he  found  in  Technical  Series   No.   11  of  this  Bureau. 
In  his  publications  the  author  has  made  no  claim  that  Bacillus  larva 

is  the  cause  of  American   foul    hrood.  hut    has   made  the  statement  that 
it  is  found  to  he  present  in  all   the   samples  of  this  disease  which  have 


1  The  author  wishes  to  acknowledge  the  assistance  "t   I'r.  K.  F.  Phillips,   in 
Charge  oi  Apiculture,  under  whose  direction  tin.-  «erk  has  been  done. 

results  have  been  reached  since  the  author  has  been  connected  with  the  apicul- 
tural  investigations  of  the  Bureau  oi  Entomolo( 


been  examined  by  him.  No  inoculation  experiments  were  made,  for 
the  reason  that  sufficient  cultures  in  suitable  condition  could  not  be 
obtained  from  any  medium  then  known. 

Since  the  media  used  in  former  investigations  are  not  suitable  for 
obtaining  cultures  for  purposes  of  inoculation,  in  taking  up  the  further 
study  it  has  been  necessary  to  devise  a  medium  which  would  be  satis- 
factory in  this  respect.  Such  a  medium  has  been  discovered,  and 
large  amounts  of  the  culture  suitable  for  experimental  inoculations  have 
been  obtained.  This  medium  is  prepared  and  used  as  follows  :  Healthy 
bee  larva?  or  young  pupa'  are  picked  from  the  comb,  crushed,  strained 
through  cheese  cloth,  diluted  with  20  to  50  times  their  volume  of  water, 
filtered  through  ordinary  filter  paper,  and  then  passed  through  an  earthen- 
ware filter  (the  Berkefeld  filter  is  satisfactory)  to  remove  any  bacteria 
which  are  present.  The  sterile  filtrate  thus  obtained  may  be  pipetted 
into  tubes  or  flasks  and  stored  until  needed.  When  Bacillus  larva  is 
to  be  isolated,  a  tube  of  the  ordinary  agar  of  the  laboratory  is  liquefied 
and  cooled  to  45°  or  50°  C.  Then  about  2  c.c.  of  the  filtrate  men- 
tioned above  is  added  to  it.  A  very  small  amount  of  the  decaying 
larva'  affected  with  American  foul  brood  is  then  added.  The  procedure 
from  this  point  is  as  usual  in  making  agar  plate  cultures;  these  plates 
are  afterward  incubated.  When  a  large  amount  of  culture  is  desired  for 
experimental  purposes  it  is  convenient  to  use  the  ordinary  agar  medium 
in  large  test  tubes  to  which  has  been  added,  as  above,  about  2  c.c.  of 
the  sterile  larva-  filtrate.  These  agar  tubes  are  then  inclined  and  the 
surface  of  the  congealed  agar  is  inoculated.  In  no  case  should  the  larva1 
or  filtrate  reach  a  high  temperature.  The  object,  of  course,  is  to  obtain 
a  medium  which  contains  the  food  constituents  which  are  afforded  the 
bacteria  in  the  living  larva?. 

Inoculation  experiments  have  been  made  by  feeding  to  a  healthy 
colony  the  scales  from  combs  which  had  contained  brood  affected  with 
American  foul  brood.  The  result  of  the  feeding  was  that  the  colony 
became  affected  b}T  disease,  the  symptoms  of  which  were  the  same  as 
those  observed  in  the  apiary  where  American  foul  brood  is  found.  Like 
symptoms  have  been  produced  by  feeding  scales  which  had  been  put 
into  ordinary  meat  bouillon,  incubated  for  twenty-four  hours,  and  then 
heated  to  65°  C.  for  twenty  minutes. 

On  microscopic  examination  of  the  decaying  larva3  dead  from  the 
disease  thus  produced  experimentally,  the  same  large  number  of  spores 
and  rods  are  seen  as  when  samples  are  examined  which  are  taken  from 
an  apiary  affected  with  American  foul  brood.  From  these  dead  larva' 
pure  cultures  of  Bacillus  larva  were  obtained  from  plates,  using  the 
new  medium  described  above.  These  experiments  show  that  by  the 
feeding  method  the  disease  may  he  produced  and  that  the  contagion  is 
found  in  the  scales.     The  second  experiment  tends  to  indicate  that  the 


cause  of  American  fool  brood  as  found  in  thi  ie  not   killed  by 

heal  .1!  ■  pplied  for  twenty  minuti 

Up  to  the  presenl  time  there  1-  no  authentic  record  of  thie  disi 
having  been  produced  by  experimental  inoculations  of  pure  cultui 

Knowing  thai  by  the  feeding  method  the  disease  may  be  produced, 
pure  cultures  of  Bacillus  larva  have  been  mixed  with  sterile  sugar 
sirup  and  fed  to  healthy  colonies  with  the  result  thai  the  die 
appeared  in  the  colonies  within  three  weeks  with  symptome  identical 
with  those  produced  by  feeding  the  Bcales  of  the  disease.  In  the  ropj 
brown  mass  of  the  decaying  larvae  in  the  disease  which  is  produced 
experimentally  by  feeding  pure  cultures  of  Bacillus  larva  there  are 
found  the  same  large  number  of  spores  and  rods  as  when  the  disease  ie 
produced  by  feeding  th>-  or  when   the  disease   is   found  in  an 

apiary.  Pure  cultures  ol  Bacillus  larva  have  been  obtained  from  the 
larva'  dead  from  the  disease  produced  experimentally  by  feeding  pure 
cultures  of  Bacillus  larva  . 

Some  European  investigators  of  brood  diseases  omil  the  symptoms, 
so  thai  it  is  impossible  to  tell  which  disease  they  are  investigating. 
Their  descriptions  of  microorganisms  also  are  entirely  too  brief.  These 
facts  have  led  to  much  confusion,  and  they  necessitate  much  additional 
work  on  the  part  of  other  investigators.  They  have  also  added  to  the 
presenl  confusion.  From  what  can  be  gained  from  their  papers,  the 
author  is  inclined  to  believe  thai  Burri  has  been  working  with  Bacillus 
larva  and  lias  Keen  referring  to  it  as  the  "bacillus  difficult  of  cultiva- 
tion;" thai  Maassen  has  been  working  with  Bacillus  larva  and  has 
been  referring  to  it  as  Bacillus  brandenburgiensis,  and  thai  von  Butte) 
Reepen  lias  referred  to  Bacillus  larva  as  "/»'.  fwrri."  It  is  hoped  thai 
this  confusion  may  soon  cease  to  exist. 

In  the  study  of  Bacillus  larva  on  this  new  medium  Borne  interesting 
additional  facts  have  been  observed  in  the  morphology  and  cultural 
characters  of  this  organism  which  will  be  given  in  a  bulletin  from  this 
Bureau  in  the  near  future.  One  fact  i-  mentioned  now  because  it  seems 
to  have  caused  one  German  investigator,  Dr.  Allien  Maassen,  to  fall 
into  error  in  the  interpretation  of  certain  findings.  This  fact  is  that 
this  specie.-.  Bacillus  larva,  produce-  a  large  number  of  gianl  whips. 
(Gianl  whips  are  at  present  believed  to  he  in  Borne  way  a  modification 
of  Qagella,  the  motile  organs  of  bacteria.)  These  gianl  whip-  appear 
in  pure  culture-  of  Bacillus  larva  and  persist  there  for  a  long  time. 
structures  which  Maassen  evidently  saw  and  reported  in  two  diiTer- 
ent  publications,  naming  them  8pirocha>ta  apis,  are  nothing  other  than 
gianl  whips  which  normally  belong  to  Bacillus  larva  and  which  an' 
formed  by  the  growth  of  Bacillus  larva  in  the  larva'  of  the  bi 

Maassen  Beems  t<>  have  no  further  evidence  that  the  structures  which 

w  are  spirochetes  than  what  could  he  gained  by  a  microscopic 

examination  of  the  remains  of  the  dead  larva'  which   had  suffered  from 


UNIVERSITY  OF  FLORIDA 


3  1262  09216  4614 

this  disease.  The  appearance  which  he  interprets  as  a  spirochete  in 
the  process  of  division  can  be  seen  in  the  giant  whips  obtained  from 
pure  cultures  of  Bacillus  larva.  These  giant  whips  are  found  in  the 
decaying  larvae  which  are  dead  from  American  foul  brood  experimentally 
produced  by  feeding  pure  cultures  of  Bacillus  larva . 

The  author  has  observed  these  structures  in  a  large  number  of  exam- 
inations of  American  foul  brood,  especially  in  the  hanging-drop  prepa- 
rations made  directly  from  the  dead  larva'.  There  is  nothing  else 
contained  in  the  dead  larva'  which  can  be  seen  that  resembles  a  spiro- 
chete, and  since  Maassen  made  no  mention  of  the  giant  whips  found 
there  so  abundantly,  it  is  quite  certain  that  he  has  made  this  mistake. 

This  preliminary  note  will  be  followed  by  a  bulletin  which  will  con- 
tain in  full  the  results  of  recent  investigations  by  others  on  the  brood 
diseases  of  bees  and  a  detailed  account  of  the  work  done  here. 

The  results  may  lie  summarized  as  follows: 

(1)  In  previous  publications  the  author  has  made  no  claim  that 
Bacillus  larva  is  the  cause  of  American  foul  brood. 

(2)  A  medium  has  been  devised  by  which  cultures  of  Bacillus  larva 
may  be  obtained  in  large  quantities  suitable  for  experimental  inocula- 
tion. This  medium  consists  of  the  sterile  filtrate  obtained  by  diluting 
and  filtering  the  crushed  bodies  of  bee  larva-  through  a  Berkefeld  or 
other  fine  filter. 

(3)  American  foul  brood  has  been  produced  by  feeding  pure  cultures 
of  Bacillus  larva  ,  and  the  symptoms  of  the  disease  are  the  same  as 
those  produced  by  feeding  the  scales  of  this  disease  and  as  those 
ol .served  in  the  apiary  where  colonies  are  affected  with  this  disease. 

(4)  The  structures  described  by  Doctor  Maassen,  of  Dahlem,  Ger- 
many, as  spirochetes  and  named  by  him  Spirocha  la  apis  are  not  spi- 
rochetes, but  normal  structures,  produced  by  the  growth  of  Bacillus 
larva.     These  are  known  in  bacteriology  as  giant  whips. 

Approved : 

W.  M.  Hays. 

Acting  Secretary  of  Agriculture. 

Washington,  D.  C,  July  15,  W<>7. 

O 


